The present invention relates to an improved process for the preparation of certain epothilone analogs, including novel intermediates, which is characterized by a significantly enhanced yield.
Epothilones are macrolide compounds that find utility in the pharmaceutical field. For example, epothilones A and B having the structures: 
epothilone A Rxe2x95x90H
epothilone B Rxe2x95x90Me
may be found to exert microtubule-stabilizing effects similar to paclitaxel (TAXOL(copyright)) and hence cytotoxic activity against rapidly proliferating cells, such as, tumor cells or other hyperproliferative cellular disease, see Hofle, G., et al., Angew. Chem. Int. Ed. Engl., Vol. 35, No.13/14, 1567-1569 (1996); WO93/10121 published May 27, 1993; and WO97/19086 published May 29, 1997.
Derivatives and analogs of epothilones A and B have been synthesized and may be used to treat a variety of cancers and other abnormal proliferative diseases. Such analogs are disclosed in Hofle et al., Id.; Nicolaou, K. C., et al., Angew. Chem. Int. Ed. Engl., Vol. 36, No. 19, 2097-2103 (1997); and Su, D.-S., et al., Angew. Chem. Int. Ed. Engl., Vol. 36, No. 19, 2093-2097 (1997).
Analogs of the epothilones that have been found to have advantageous activity are represented by the following structure 
wherein Q, and R1 through R6 have the meanings given herein below. An improved synthesis for these analogs involving novel intermediates is provided in accordance with the present invention.
The present invention is directed to a process for the preparation of compounds represented by formulas I and II wherein Q, Z, and R1 through R6 are as defined below. 
The compounds represented by formula I are novel intermediates for the preparation of epothilone analogs that are useful in the treatment of a variety of cancers and other abnormal proliferative diseases. Compounds represented by formula I may be utilized to prepare epothilone analogs represented by formula II which are useful as anticancer agents.
The process of the present invention provides an advantageous synthesis for the compounds represented by formula II including the preparation of novel ring opened epothilone intermediate compounds represented by formula I. 
As used in formulas I and II, and throughout the specification, the meaning of the symbol Q is: 
M is selected from the group consisting of oxygen, sulfur, NR8, and CR9R10;
Z is selected from the group consisting of 
R1-R5, R7, and R11-R15 are selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl and heterocyclo, and wherein R1 and R2 are alkyl, they can be joined to form a cycloalkyl;
R6 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, heterocyclo and substituted heterocyclo;
R8 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, R11Cxe2x95x90O, R12OCxe2x95x90O and R13SO2;
R9 and R10 are selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, aryl, heterocyclo, hydroxy, R14Cxe2x95x90O, and R15OCxe2x95x90O; and
R16, R17, and R18 are independently selected from the group consisting of alkyl, aryl, and aralkyl.
The process of the present invention is advantageous in that only two steps are required to prepare the epothilone analogs from the epothilone starting material, for example, epothilone B. Two further distinct advantages of the process of the present invention are that the yields of crystallized compounds represented by formula II are significantly higher than those previously realized utilizing the free acid of the compound represented by formula I as the intermediate compound, and the fact that the preparation of the intermediate is amendable to being carried out in one step. A further advantage of this process is that it can progress from the epothilone starting material to the epothilone represented by formula II without the need to isolate and purify an intermediate. Those skilled in the art will immediately recognize the economic benefits of such a process.
Definitions
The following are definitions of various terms used herein to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
The term xe2x80x9cepothilonexe2x80x9d, as used herein, denotes compounds containing an epothilone core and a side chain group as defined herein. The term xe2x80x9cepothilone corexe2x80x9d, as used herein, denotes a moiety containing the core structure (with the numbering of ring system positions used herein shown): 
wherein the substituents are as defined herein and where
X is selected from the group consisting of Cxe2x95x90O, CH2 and CHOR19;
B1 and B2 are selected from the group consisting of OR20 and OCOR21;
R19 and R20 are selected from the group consisting of hydrogen, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl, and dialkylarylsilyl; and
R21 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and heterocyclo.
The term xe2x80x9cside chain groupxe2x80x9d refers to substituent G as defined by the following formula
Ymxe2x80x94Axe2x80x94
where
A is optionally substituted alkenyl;
Y is an optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring; and
m is zero or 1.
The term xe2x80x9calkylxe2x80x9d refers to optionally substituted straight- or branched-chain saturated hydrocarbon groups having from 1 to 20 carbon atoms, preferably from 1 to 7 carbon atoms. The expression xe2x80x9clower alkylxe2x80x9d refers to optionally substituted alkyl groups having from 1 to 4 carbon atoms.
The term xe2x80x9csubstituted alkylxe2x80x9d refers to an alkyl group substituted by, for example, one to four substituents, such as, halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyoxy, heterocylooxy, oxo, alkanoyl, aryl, aryloxy, aralkyl, alkanoyloxy, amino, alkylamino, arylamino, aralkylamino, cycloalkylamino, heterocycloamino, disubstituted amino in which the two substituents on the amino group are selected from alkyl, aryl, aralkyl, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, aralkylthio, cycloalkylthio, heterocyclothio, alkylthiono, arylthiono, aralkylthiono, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, sulfonamido (e.g. SO2NH2), substituted sulfonamido, nitro, cyano, carboxy, carbamyl (e.g. CONH2), substituted carbamyl (e.g. CONH alkyl, CONH aryl, CONH aralkyl or instances where there are two substituents on the nitrogen selected from alkyl, aryl or aralkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino and heterocyclos, such as, indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Wherein, as noted above, the substituents themselves are further substituted, such further substituents are selected from the group consisting of halogen, alkyl, alkoxy, aryl and aralkyl. The definitions given herein for alkyl and substituted alkyl apply as well to the alkyl portion of alkoxy groups.
The term xe2x80x9calkenylxe2x80x9d refers to optionally substituted unsaturated aliphatic hydrocarbon groups having from one to nine carbons and one or more double bonds. Substituents may include one or more substituent groups as described above for substituted alkyl.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to fluorine, chlorine, bromine and iodine.
The term xe2x80x9cring systemxe2x80x9d refers to an optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring. Exemplary ring systems include, but are not limited to, an aryl or a partially or filly unsaturated heterocyclic ring system, which may be optionally substituted.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or bicyclic aromatic hydrocarbon groups having from 6 to 12 carbon atoms in the ring portion, for example, phenyl, naphthyl, biphenyl and diphenyl groups, each of which may be substituted.
The term xe2x80x9caralkylxe2x80x9d refers to an aryl group bonded to a larger entity through an alkyl group, such as benzyl.
The term xe2x80x9csubstituted arylxe2x80x9d refers to an aryl group substituted by, for example, one to four substituents such as alkyl; substituted alkyl, halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, aralkylamino, cycloalkylamino, heterocycloamino, alkanoylamino, thiol, alkylthio, cycloalkylthio, heterocyclothio, ureido, nitro, cyano, carboxy, carboxyalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono, alkysulfonyl, sulfonamido, aryloxy and the like. The substituent may be further substituted by one or more members selected from the group consisting of halo, hydroxy, alkyl, alkoxy, aryl, substituted alkyl, substituted aryl and aralkyl.
The term xe2x80x9ccycloalkylxe2x80x9d refers to optionally substituted saturated cyclic hydrocarbon ring systems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring, which may be further fused with an unsaturated C3-C7 carbocyclic ring. Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and adamantyl. Exemplary substituents include one or more alkyl groups as described above, or one or more of the groups described above as substituents for alkyl groups.
The terms xe2x80x9cheterocyclexe2x80x9d, xe2x80x9cheterocyclicxe2x80x9d and xe2x80x9cheterocycloxe2x80x9d refer to an optionally substituted, unsaturated, partially saturated, or fully saturated, aromatic or nonaromatic cyclic group, for example, which is a 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring system, which has at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms, where the nitrogen and sulfur heteroatoms may also optionally be oxidized and the nitrogen heteroatoms may also optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom.
Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxazepinyl, azepinyl, 4-piperidonyl, pyridyl, N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothienyl, dioxanyl, isothiazolidinyl, thietanyl, thiiranyl, triazinyl, and triazolyl, and the like.
Exemplary bicyclic heterocyclic groups include benzothiazolyl, benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl, quinolinyl-N-oxide, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,1-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), benzisothiazolyl, benzisoxazolyl, benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzotriazolyl, benzpyrazolyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydrobenzopyranyl, indolinyl, isochromanyl, isoindolinyl, naphthyridinyl, phthalazinyl, piperonyl, purinyl, pyridopyridyl, quinazolinyl, tetrahydroquinolinyl, thienofuryl, thienopyridyl, thienothienyl, and the like.
Exemplary substituents for the terms xe2x80x9cring system,xe2x80x9d xe2x80x9cheterocycle,xe2x80x9d xe2x80x9cheterocyclic,xe2x80x9d and xe2x80x9cheterocycloxe2x80x9d include one or more substituent groups as described above for substituted alkyl or substituted aryl, and smaller heterocyclos, such as, epoxides, aziridines and the like.
The term xe2x80x9calkanoylxe2x80x9d refers to xe2x80x94C(O)-alkyl.
The term xe2x80x9csubstituted alkanoylxe2x80x9d refers to xe2x80x94C(O)-substituted alkyl.
The term xe2x80x9cheteroatomsxe2x80x9d shall include oxygen, sulfur and nitrogen.
The compounds represented by formula II form salts with a variety of organic and inorganic acids. Such salts include those formed with hydrogen chloride, hydrogen bromide, methanesulfonic acid, hydroxyethanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid and various others as are recognized by those of ordinary skill in the art of pharmaceutical compounding. Such salts are formed by reacting a compound represented by formula II in an equivalent amount of the acid in a medium in which the salt precipitates or in an aqueous medium followed by evaporation.
In addition, zwitterions (xe2x80x9cinner saltsxe2x80x9d) can be formed and are included within the term salts as used herein.
The compounds represented by formulae I and II above may exist as multiple optical, geometric, and stereoisomers. While the compounds shown herein are depicted for one optical orientation, included within the present invention are all isomers and mixtures thereof.
Use and Utility
The invention is a process by which compounds represented by formula II above that are microtubule-stabilizing agents are produced. The compounds, and thus the process, are useful in the treatment of a variety of cancers and other proliferative diseases including, but not limited to, the following;
carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma;
hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;
other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma;
tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas;
tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and
other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma.
The compounds produced by the invention as represented by formula II above will also inhibit angiogenesis, thereby affecting the growth of tumors and providing treatment of tumors and tumor-related disorders. Such anti-angiogenesis properties of the compounds represented by formula II will also be useful in the treatment of other conditions responsive to anti-angiogenesis agents including, but not limited to, certain forms of blindness related to retinal vascularization, arthritis, especially inflammatory arthritis, multiple sclerosis, restinosis and psoriasis.
Compounds produced by the invention as represented by formula II will induce or inhibit apoptosis, a physiological cell death process critical for normal development and homeostasis. Alterations of apoptotic pathways contribute to the pathogenesis of a variety of human diseases. Compounds represented by formula II, as modulators of apoptosis, will be useful in the treatment of a variety of human diseases with aberrations in apoptosis including, but not limited to, cancer and precancerous lesions, immune response related diseases, viral infections, degenerative diseases of the musculoskeletal system and kidney disease.
Without wishing to be bound to any mechanism or morphology, the compounds produced by the invention as represented by formula II may also be used to treat conditions other than cancer or other proliferative diseases. Such conditions include, but are not limited to viral infections such as herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus; autoimmune diseases such as systemic lupus erythematosus, immune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases and autoimmune diabetes mellitus; neurodegenerative disorders such as Alzheimer""s disease, AIDS-related dementia, Parkinson""s disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration; AIDS; myelodysplastic syndromes; aplastic anemia; ischemic injury associated myocardial infarctions; stroke and reperfusion injury; restenosis; arrhythmia; atherosclerosis; toxin-induced or alcohol induced liver diseases; hematological diseases such as chronic anemia and aplastic anemia; degenerative diseases of the musculoskeletal system such as osteoporosis and arthritis; aspirin-sensitive rhinosinusitis; cystic fibrosis; multiple sclerosis; kidney diseases; and cancer pain.
General Methods of Preparation
The novel open-ring intermediates represented by formula I can be prepared from an epothilone starting material represented by formula III in Scheme 1 wherein Q, Z, and R1 through R6 are as defined above. The epothilone starting materials represented by formula III are known compounds, see, for example, Hofle, G., et al., Angew. Chem. Int. Ed. Engl., Vol. 35, No.13/14, 1567-1569 (1996); WO93/10121 published May 27, 1993; and WO97/19086 published May 29, 1997; Nicolaou, K. C., et al., Angew Chem. Int. Ed. Engl., Vol. 36, No. 19, 2097-2103 (1997); and Su, D.-S., et al., Angew Chem. Int. Ed. Engl., Vol. 36, No. 19, 2093-2097 (1997).
As illustrated in Scheme 1, the epothilone starting material III is reacted with a suitable azide donor agent and a reducing agent in the presence of a phase transfer catalyst and a palladium catalyst under mildly acidic conditions, i.e. a pH not below about 5.5, preferably from pH 6.0 to 6.5, most preferably about 6.5, in a suitable mixed solvent system comprising water and an organic solvent such as THF, DMF and the like. The reaction is conducted at ambient temperature for an extended period, e.g. in excess of twelve hours.
The epothilone starting material for this invention can be any epothilone comprising an epothilone core and side chain as defined herein. Preferably the starting material is a compound represented by formula III in Scheme 1. 
Suitable azide donor agents for this reaction include metal azides, for example lithium or sodium azide, tetraalkylammonium azides, for example, tetrabutylammonium azide, trialkylsilyl azides, for example trimethylsilyl azide, and the like, Preferred azide donors are sodium azide and tetrabutyl ammonium azide. An especially preferred azide donor is tetrabutylammonium azide.
Suitable reducing agents are trialkylphosphine, triarylphosphine, tri(alkyl/aryl)phosphine, trialkylarsine, triarylarsine, tri(alkyl/aryl)arsine and mixtures thereof Preferred reducing agents are trimethyl phosphine, triethyl phosphine, tributyl phosphine, triphenyl phosphine, and tripropyl phosphine. An especially preferred reducing agent is trimethyl phosphine (PME3).
Suitable phase transfer catalysts or agents may include any quaternary onium salt and their corresponding anions. Suitable phase transfer agents include tetraalkylonium, tetrararylonium, tetraaralkylonium, and any combination of these types of onium substituents. More specifically the phase transfer catalyst may include tetraalkylammonium halides such as tetrabutylammonium chloride or benzyltriethylammonium chloride. An especially preferred phase transfer agent is tetrabutylammonium chloride. The onium substituent may be ammonium, phosphonium, or arsonium. Exemplary anions for these quartenary salts include, but are not limited to, halides, hydroxyl, cyano, phosphate, sulfate and the like. Other suitable phase transfer catalysts or agents are described in Yuri Goldberg, Phase Transfer Catalysis, Gordon and Breach Science Publishers, 1992, Chapter 1 and the references cited therein, the full text of which is incorporated herein by reference.
The palladium catalyst for the reaction shown in Scheme 1 may be, for example, palladium acetate, palladium chloride, palladium tetrakis-(triphenyl-phosphine), palladium tetrakis-(triphenylarsine), tris-(dibenzylideneacetone)-dipalladium(0)chloroform adduct (Pd2(dba)3.CHCl3 and the like. A preferred catalyst is tris-(dibenzylideneacetone)-dipalladium(0)chloroform adduct (Pd2(dba)3.CHCl3). Tris-(dibenzylideneacetone)-dipalladium is also a useful catalyst in the reaction illustrated in Scheme 1. The chemistry of the palladium catalysts is known, see for example, I. J. Tsuji, Palladium Reagents and Catalysts: Innovations in Organic Synthesis, New York, Wiley and Sons, 1995, the full text of which is incorporated herein by reference.
Suitable buffering agents to maintain the pH within the desired range include a mild acid or acidic salt, such as acetic acid, sodium biphosphate and, preferably, ammonium chloride.
As shown in Scheme 2, epothilone analogs represented by formula II are prepared from the novel open-ring intermediates represented by formula I by macrolactamization utilizing a suitable macrolactamization or coupling agent in a mixed organic solvent system, such as THF/DMF. 
Macrolactamization agents for the reaction include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), or EDCI in combination with 1-hydroxy-7-azabenzotriazole (HOAT) or 1-hydroxy-7-benzotriazole hydrate (HOBT), other carbodiimides such as dicyclohexylcarbodiimide and diisopropylcarbodiimide, O-benzotriazol-1-yl-N,N, Nxe2x80x2,Nxe2x80x2-bis(tetramethylene)uronium hexafluorophosphate (HBTu/DMAP), O-(7-azabenzotriazol)-1-yl-N,N,Nxe2x80x2,Nxe2x80x2-bis(tetramethylene)uronium hexafluorophosphate (HATu/DMAP), benzotriazole-1-yloxy-tris(bimethylamino)phosphonium hexafluorophosphate (BOP), N,N-dimethyl-4-aminopyridine (DMAP), K2CO3, diisopropylamine, triethylamine and the like. A preferred macrolactamization agent includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) in combination with 1-hydroxy-7-benzotriazole (HOBT). Examples of other suitable macrolactamization agents can be found in J. M. Humphrey and A. R. Chamberlin, Chem. Rev. 97, 2243-2266, (1997), the full text of which is incorporated herein by reference.
The cyclization reaction as shown in Scheme 2 is carried out in the cold, i.e. a temperature of from about 0xc2x0 C. to about xe2x88x9220xc2x0 C., preferably from about xe2x88x925xc2x0 C. to xe2x88x9210xc2x0 C.
The reaction of Scheme 2 is carried out in mildly alkaline conditions with a mild base such as K2CO3, triethylamine, diisopropylamine and the like, preferably with K2CO3, to inhibit the production of any unwanted by-products.
Scheme 3 below illustrates a preferred embodiment of the invention. The synthesis of the compounds represented by formula II from the starting epothilone material, epothilone B represented by formula III, is sequentially reacted without isolation of the novel intermediate represented by formula I as illustrated. 
It has been found in accordance with the present invention that the compounds represented by formula II can be prepared in significantly improved yields in comparison to prior methods. Typically, the instant process produces about a three fold increase in yield.
All references cited herein are incorporated by reference as if set forth at length herein.